1. Field of the Invention
The present invention relates to gene and amino acid sequences encoding DNA polymerase III holoenzyme subunits and structural genes from thermophilic organisms. In particular, the present invention provides DNA polymerase III holoenzyme subunits and accessory proteins of T. thermophilus. The present invention also provides antibodies and other reagents useful to identify DNA Polymerase III molecules.
2. Background Art
Bacterial cells contain three types of DNA polymerases termed polymerase I, II and III. DNA polymerase III (pol III) is responsible for the replication of the majority of the chromosome. Pol III is referred to as a replicative polymerase; replicative polymerases are rapid and highly processive enzymes. Pol I and II are referred to as non-replicative polymerases although both enzymes appear to have roles in replication. DNA polymerase I is the most abundant polymerase and is responsible for some types of DNA repair, including a repair-like reaction that permits the joining of Okazaki fragments during DNA replication. Pol I is essential for the repair of DNA damage induced by UV irradiation and radiomimetic drugs. Pol II is thought to play a role in repairing DNA damage which induces the SOS response and in mutants which lack both pol I and III, pol II repairs UV-induced lesions. Pol I and II are monomeric polymerases while pol III comprises a multisubunit complex.
In E. coli, pol III comprises the catalytic core of the E. coli replicase. In E. coli, there are approximately 400 copies of DNA polymerase I per cell, but only 10-20 copies of pol III (Komberg and Baker, DNA Replication, 2d ed., W. H. Freeman and Company, [1992], pp. 167; and Wu et al. J. Biol. Chem., 259:12117-12122 [1984]). The low abundance of pol III and its relatively feeble activity on gapped DNA templates typically used as a general replication assays delayed its discovery until the availability of mutants defective in DNA polymerase I (Kornberg and Gefter, J. Biol. Chem., 47:5369-5375 [1972]).
The catalytic subunit of pol III is distinguished as a component of E. coli major replicative complex, apparently not by its intrinsic catalytic activity, but by its ability to interact with other replication proteins at the fork. These interactions confer upon the enzyme enormous processivity. Once the DNA polymerase III holoenzyme associates with primed DNA, it does not dissociate for over 40 minutesxe2x80x94the time required for the synthesis of the entire 4 Mb E. coli chromosome (McHenry, Ann. Rev. Biochem., 57:519-550 [1988]). Studies in coupled rolling circle models of the replication fork suggest the enzyme can synthesize DNA 150 kb or longer without dissociation in vitro (Mok and Marians, J. Biol. Chem., 262:16644-16654 [1987]; Wu et al., J. Biol. Chem., 267:4030-4044 [1992]). The essential interaction required for this high processivity is an interaction between the xcex1 catalytic subunit and a dimer of xcex2, a sliding clamp processivity factor that encircles the DNA template like a bracelet, permitting it to rapidly slide along with the associated polymerase, but preventing it from falling off (LaDuca et al., J. Biol. Chem., 261:7550-7557 [1986]; Kong et al., Cell 69:425-437 [1992]). The xcex2-xcex1 association apparently retains the polymerase on the template during transient thermal fluctuations when it might otherwise dissociate.
The xcex22 bracelet cannot spontaneously associate with high molecular weight DNA, it requires a multiprotein DnaX-complex to open and close it around DNA using the energy of ATP hydrolysis (Wickner, Proc. Natl. Acad. Sci. USA 73:35411-3515 [1976]; Naktinis et al., J. Biol. Chem., 270:13358-13365 [1985]; and Dallmann et al., J. Biol. Chem., 270:29555-29562 [1995]). In E. coli, the dnaX gene encodes two proteins, xcfx84 and xcex3. xcex3 is generated by a programmed ribosomal frameshifting mechanism five-sevenths of the way through dnaX mRNA, placing the ribosome in a xe2x88x921 reading frame where it immediately encounters a stop codon (Flower and McHenry Proc. Natl. Acad. Sci. USA 87:3713-3717 [1990]; Blinkowa and Walker, Nucl. Acids Res., 18:1725-1729 [1990]; and Tsuchihashi and Kornberg, Proc. Nati. Acad. Sci. USA 87:2516-2520 [1990]). In E. coli, the DnaX-complex has the stoichiometry xcex32xcfx842xcex41xcex4xe2x80x21 "khgr"1"igr"1 (Dallmann and McHenry, J. Biol. Chem., 270:29563-29569 [1995]). The xcfx84 protein contains an additional carboxyl-terminal domain that interacts tightly with the polymerase, holding two polymerases together in one complex that can coordinately replicate the leading and lagging strand of the replication fork simultaneously (McHenry, J. Biol. Chem., 257:2657-2663 [1982]; Studwell and O""Donnell, Biol. Chem., 266:19833-19841 [1991]; McHenry, Ann. Rev. Biochem. 57:519-550 [1988]).
Conservation of a frameshifting mechanism to generate related ATPases is significant in that, by analogy to E. coli, can both assemble a processivity factor onto primed DNA. In E. coli, ribosomes frameshift at the sequence A AAA AAG into a xe2x88x921 frame where the lysine UUU anticodon tRNA can base pair with 6As before elongating (Flower and McHenry, Proc. Natl. Acad. Sci. USA 87:3713-3717 [1990]; Blinkowa and Walker, Nucl. Acids Res., 18:1725-1729 [1990]; and Tsuchihashi and Kornberg, Proc. Natl. Acad. Sci. USA 87:2516-2520 [1990]).
Pol IIIs are apparently conserved throughout mesophilic eubacteria. In addition to E. coli and related proteobacteria, the enzyme has been purified from the firmicute Bacillus subtilis (Low et al., J. Biol. Chem., 251:1311-1325 [1976]; Hammond and Brown [1992]). With the proliferation of bacterial genomes sequenced, by inference from DNA sequence, pol III exists in organisms as widely divergent as Caulobacter, Mycobacteria, Mycoplasma, B. subtilis and Synechocystis. The existence of dnaX and dnaN (structural gene for xcex2) is also apparent in these organisms. These general replication mechanisms are conserved even more broadly in biology. Although eukaryotes do not contain polymerases homologous to pol III, eukaryotes contain special polymerases devoted to chromosomal replication and xcex2-like processivity factors (PCNA) and DnaX-like ATPases (RFC, Activator I) that assemble these processivity factors on DNA (Yoder and Burgers, J. Biol. Chem., 266:22689-22697 [1991]; Brush and Stillman, Meth. Enzymol., 262:522-548 [1995]; Uhlmann et al., Proc. Nati. Acad. Sci. USA 93:6521-6526 [1996]).
Helicases serve a variety of functions in DNA metabolism. Cellular (E. coli dnaB, priA, and rep proteins), phage (T4 gene 41 and dda proteins; T7 gene 4 protein), and viral (SV40 T antigen; HSV-1 UL5/UL52 complex and UL9 protein) helicases are involved in the initiation of replication, by unwinding DNA so that other proteins of the replication complex can assemble on the ssDNA. These proteins also participate in the elongation phase of replication, by unwinding the duplex DNA ahead of this complex to provide the required template. Other helicases (e.g., the E. coli recBCD and recQ proteins) are implicated in recombination by genetic criteria. Another class of helicases includes the E. coli uvrAB and uvrD. These helicases act in nucleotide excision repair or methyl-directed mismatch repair during both pre-incision (recognition of DNA damage or alteration) and post-incision (displacement of damaged fragment) steps. See, for example, U.S. Pat. No. 5,747,247.
DNA mispairing can occur in vivo and is recognized and corrected by repair proteins. Mismatch repair has been studied most intensively in E. coli, Salmonella typhimurium, and S. pneumoniae. The MutS, MutH and MutL proteins of E. coli are involved in the repair of DNA mismatches, as is the product of the uvrD gene in E. coli, helicase II. See, for example, U.S. Pat. No. 5,750,335.
The best defined mismatch repair pathway is the E.coli MutHLS pathway that promotes a long-patch (approximately 3 Kb) excision repair reaction which is dependent on the mutH, mutL, mutS and mutU (uvrD) gene products. The MutHLS pathway appears to be the most active mismatch repair pathway in E.coli and is known to both increase the fidelity of DNA replication and to act on recombination intermediates containing mispaired bases. The system has been reconstituted in vitro, and requires the mutH, mutL, mutS and uvrD (helicase II) proteins along with DNA polymerase III holoenzyme, DNA ligase, single-stranded DNA binding protein (SSB) and one of the single-stranded DNA exonucleases, Exo I, Exo VII or RecJ. A similar pathway in yeast includes the yeast MSH2 gene and two mutL-like genes referred to as PMS1 and MLH1. See, for example, U.S. Pat. No. 6,191,268.
The E. coli bacterial Uvr proteins are capable of excising damaged DNA sites caused by a broad spectrum of chemical agents that distort the backbone geometry of the DNA double helix. As a result, if the DNA were damaged by chemicals in the environmental sample, the Uvr proteins will cleave and excise the damaged region. Subsequent resynthesis by DNA polymerase I will incorporate labeled or unlabeled nucleotides into the DNA. See, for example, U.S. Pat. No. 6,060,288.
Replication of the lagging strand of DNA is mediated by a multiprotein complex composed of proteins priA, dnaT, dnaB, dnaC, and dnaG. This complex is referred to as a primosome. Purified priA has ATPase, helicase, translocase, and primosome assembly activities. This gene may be essential in recombination and DNA repair since it binds to D-loops, interacts with recG and has helicase activity. The 3xe2x80x2-5xe2x80x2 DNA helicase activity of priA inhibits recombination. See, for example, U.S. Pat. No. 6,146,846.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (uvrD helicase) 68.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (DNA-G Primase) 72.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (priA helicase) 76.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (delta subunit) 10.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (delta prime subunit) 17.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (beta subunit) 23.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (ssb protein) 32.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (epsilon-1, dnaQ-1) 37.
The invention is directed to an isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (epsilon-2, dnaQ-2) 82.
The invention is directed to a method of producing a polypeptide encoded by a nucleotide sequence, wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of one of SEQ ID NOS: 68, 72, 76, 10, 17, 23, 32, 37, and 82, comprising culturing a host cell comprising said nucleotide sequence under conditions such that said polypeptide is expressed, and recovering said polypeptide.
The invention is directed to a method of synthesizing DNA which comprises utilizing one or more polypeptides, said one or more polypeptides comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 68, 72, 76, 10, 17, 23, 32, 37 and 82.
Further objects and advantages of the present invention will be clear from the description that follows.